Cytochrome P450 Inhibition, LC-MS/MS Detection (IC50 Determination)
Assay System:
  • Human liver microsomes, pooled from 50 donors.
  • Inhibition potential of test compound measured upon incubation with human liver microsomes at different compound concentrations. Reversible inhibition is determined by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.

Assay Conditions:

Test Compound Concentration   Eight concentrations ranging from 0.05 µM to 100 µM
Percent Organic Solvent in Incubation   2% acetonitrile
CYP Isoforms Available for Testing   CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
Incubation Time   10-20 minutes (depending on CYP isoform)
pH   7.4
Temperature   37 °C
Analysis   LC-MS/MS
Number of Repeats   2

Deliverable: IC50 of test compound and positive control compound.

Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.
Probe Substrate
Phenacetin
Bupropion
Amodiaquine
Tolbutamide
S-Mephenytoin
Dextromethorphan
Testosterone
Midazolam
CYP Isoform
1A2
2B6
2C8
2C9
2C19
2D6
3A4
3A4
Monitored Metabolite
Acetaminophen
OH Bupropion
Desethylamodiaquine
4’-OH Tolbutamide
4’-OH Mephenytoin
Dextrorphan-D-Tartrate
6β-OH Testosterone
1’-OH Midazolam
Positive Control Inhibitor
Furafylline
Ticlopidine HCl
Montelukast
Sulfaphenazole
(+)-N-3-Benzylnirvanol
Quinidine
Ketoconazole
Ketoconazole